1. Field of the Invention
The present invention relates to cyclin-derived peptides for use in the improved treatment of cancer in a patient, particularly in the form of a combination therapy using a vaccine. Other aspects relate to the use of the peptides or a combination thereof as a diagnostic tool.
2. Background of the Invention
Stimulation of an immune response is dependent upon the presence of antigens recognized as foreign by the host immune system. The discovery of the existence of tumor associated antigens has now raised the possibility of using a host's immune system to intervene in tumor growth. Various mechanisms of harnessing both the humoral and cellular arms of the immune system are currently being explored for cancer immunotherapy.
Certain elements of the cellular immune response are capable of specifically recognizing and destroying tumor cells. The isolation of cytotoxic T-cells (CTL) from tumor-infiltrating cell populations or from peripheral blood suggests that these cells play an important role in natural immune defenses against cancer (Cheever et al., Annals N.Y. Acad. Sci. 1993 690:101-112; Zeh H J, Perry-Lalley D, Dudley M E, Rosenberg S A, Yang J C; J. Immunol. 1999, 162(2):989-94; High avidity CTLs for two self-antigens demonstrate superior in vitro and in vivo antitumor efficacy.). CD8-positive T-cells (TCD8+) in particular, which recognize Class I molecules of the major histocompatibility complex (MHC)-bearing peptides of usually 8 to 10 amino acid residues derived from proteins or defective ribosomal products (DRiPS) (Schubert U, Anton L C, Gibbs J, Norbury C C, Yewdell J W, Bennink J R.; Rapid degradation of a large fraction of newly synthesized proteins by proteasomes; Nature 2000; 404(6779):770-774) located in the cytosol, play an important role in this response. The MHC-molecules of the human are also designated as human leukocyte-antigens (HLA).
There are two classes of MHC-molecules: MHC class I molecules that can be found on most cells having a nucleus which present peptides that result from proteolytic cleavage of endogenous proteins, DRIPS, and larger peptides. MHC class II molecules can be found predominantly on professional antigen presenting cells (APCs), and present peptides of exogenous proteins that are taken up by APCs during the course of endocytosis, and are subsequently processed (Cresswell P. Annu Rev. Immunol. 1994; 12:259-93). Complexes of peptide and MHC class I molecules are recognized by CD8-positive cytotoxic T-lymphocytes bearing the appropriate TCR, complexes of peptide and MHC class II molecules are recognized by CD4-positive-helper-T-cells bearing the appropriate TCR. It is well known that the TCR, the peptide and the MHC are thereby abundant in a stoichiometric amount of 1:1:1.
CD4-positive helper T-cells play an important role in orchestrating the effector functions of anti-tumor T-cell responses and for this reason the identification of CD4-positive T-cell epitopes derived from tumor associated antigens (TAA) may be of great importance for the development of pharmaceutical products for triggering anti-tumor immune responses (Kobayashi, H., R. Omiya, M. Ruiz, E. Huarte, P. Sarobe, J. J. Lasarte, M. Herraiz, B. Sangro, J. Prieto, F. Borras-Cuesta, and E. Celis. 2002. Identification of an antigenic epitope for helper T lymphocytes from carcinoembryonic antigen. Clin. Cancer Res. 8:3219-3225., Gnjatic, S., D. Atanackovic, E. Jager, M. Matsuo, A. Selvakumar, N. K. Altorki, R. G. Maki, B. Dupont, G. Ritter, Y. T. Chen, A. Knuth, and L. J. Old. 2003. Survey of naturally occurring CD4+ T-cell responses against NY-ESO-1 in cancer patients: Correlation with antibody responses. Proc. Natl. Acad. Sci. U.S.A. 100(15):8862-7) CD4+ T cells can lead to locally increased levels of IFNγ (Qin Z, Schwartzkopff J, Pradera F, Kammertoens T, Seliger B, Pircher H, Blankenstein T; A critical requirement of interferon gamma-mediated angiostasis for tumor rejection by CD8+ T cells; Cancer Res. 2003 J; 63(14):4095-4100).
In the absence of inflammation, expression of MHC class II molecules is mainly restricted to cells of the immune system, especially professional antigen-presenting cells (APC), e.g., monocytes, monocyte-derived cells, macrophages, dendritic cells. In tumor patients, cells of the tumor have surprisingly been found to express MHC class II molecules (Dengjel J, Nastke M D, Gouttefangeas C, Gitsioudis G, Schoor O, Altenberend F, Müller M, Krämer B, Missiou A, Sauter M, Hennenlotter J, Wernet D, Stenzl A, Rammensee H G, Klingel K, Stevanović S.; Unexpected abundance of HLA class II presented peptides in primary renal cell carcinomas; Clin Cancer Res. 2006; 12:4163-4170).
Since the constitutive expression of HLA class II molecules is usually limited to cells of the immune system (Mach, B., V. Steimle, E. Martinez-Soria, and W. Reith. 1996. Regulation of MHC class II genes: lessons from a disease. Annu Rev. Immunol. 14:301-331), the possibility of isolating class II peptides directly from primary tumors was not considered possible. However, Dengjel et al. were recently successful in identifying a number of MHC Class II epitopes directly from tumors (EP 04 023 546.7, EP 05 019 254.1; Dengjel J, Nastke M D, Gouttefangeas C, Gitsioudis G, Schoor O, Altenberend F, Müller M, Krämer B, Missiou A, Sauter M, Hennenlotter J, Wernet D, Stenzl A, Rammensee HG, Klingel K, Stevanović S.; Unexpected abundance of HLA class II presented peptides in primary renal cell carcinomas; Clin Cancer Res. 2006; 12:4163-4170).
For a peptide to trigger (elicit) a cellular immune response, it must bind to an MHC-molecule. This process is dependent on the allele of the MHC-molecule and of the amino acid sequence of the peptide. MHC-class-1-binding peptides are usually 8-10 amino acid residues in length and usually contain two conserved residues (“anchors”) in their sequence that interact with the corresponding binding groove of the MHC-molecule. In this way each MHC allele has a “binding motif” determining which peptides can bind specifically to the binding groove (Rammensee H. G., Bachmann J. and Stevanovic, S; MHC Ligands and Peptide Motifs, Chapman & Hall 1998). In the MHC class I dependent immune reaction, peptides not only have to be able to bind to certain MHC class I molecules expressed by tumor cells, they also have to be recognized by T-cells bearing specific T-cell receptors (TCR).
The antigens that are recognized by the tumor specific cytotoxic T-lymphocytes, that is, their epitopes, can be molecules derived from all protein classes, such as enzymes, receptors, transcription factors, etc. which are up-regulated in cells of the respective tumor. Furthermore, tumor associated antigens, for example, can also be unique to tumor cells, for example as products of mutated genes or from alternative open reading frames (ORFs), or from protein splicing (Vigneron N, Stroobant V, Chapiro J, Ooms A, Degiovanni G, Morel S, van der Bruggen P, Boon T, Van den Eynde B J. An antigenic peptide produced by peptide splicing in the proteasome, Science 2004 Apr. 23; 304 (5670):587-90.). Another important class of tumor associated antigens are tissue-specific antigens, such as CT (“cancer testis”)-antigens that are expressed in different kinds of tumors and in healthy tissue of the testis. Basically, any peptide able to bind a MHC molecule may function as a T-cell epitope. A prerequisite for the induction of an in vitro or in vivo T-cell-response is the presence of a T-cell with a corresponding TCR and the absence of immunological tolerance for this particular epitope.
Therefore, TAAs are a starting point for the development of a tumor vaccine. The methods for identifying and characterizing the TAAs are based on the use of CTL that can be isolated from patients or healthy subjects, or they are based on the generation of differential transcription profiles or differential peptide expression patterns between tumors and normal tissues (Lemmel C., Weik S., Eberle U., Dengjel J., Kratt T., Becker H. D., Rammensee H. G., Stevanovic S, Nat. Biotechnol. 2004 April; 22(4):450-4, T. Weinschenk, C. Gouttefangeas, M. Schirle, F. Obermayr, S. Walter, O, Schoor, R. Kurek, W. Loeser, K. H. Bichler, D. Wernet, S. Stevanovic, and H. G. Rammensee. Integrated functional genomics approach for the design of patient-individual antitumor vaccines. Cancer Res. 62 (20):5818-5827, 2002.).
However, the identification of genes overexpressed in tumor tissues or human tumor cell lines, or selectively expressed in such tissues or cell lines, does not provide precise information as to the use of the antigens transcribed from these genes in an immune therapy. This is because only an individual subpopulation of epitopes of these antigens are suitable for such an application since a T-cell with a corresponding TCR has to be present and immunological tolerance for this particular epitope needs to be absent or minimal. It is therefore important to select only those peptides from overexpressed or selectively expressed proteins that are presented in connection with MHC molecules against which a functional T-cell can be found. Such a functional T-cell is defined as a T-cell that upon stimulation with a specific antigen can be clonally expanded and is able to execute effector functions (“effector T-cell”).
T-helper cells play an important role in orchestrating the effector function of CTLs in anti-tumor immunity. T-helper cell epitopes that trigger a T-helper cell response of the TH1 type support effector functions of CD8-positive killer T-cells, which include cytotoxic functions directed against tumor cells displaying tumor-associated peptide/MHC complexes on their cell surfaces. In this way tumor-associated T-helper cell epitopes, alone or in combination with other tumor-associated peptides, can serve as active pharmaceutical ingredients of vaccine compositions which stimulate anti-tumor immune responses.
Since both types of response, CD8 and CD4 dependent, contribute jointly and synergistically to the anti-tumor effect, the identification and characterization of tumor-associated antigens recognized by either CD8+ CTLs (ligand: MHC class I molecule+peptide epitope) or by CD4-positive CTLs (ligand: MHC class II molecule+peptide epitope) is important in the development of tumor vaccines.
Cyclin D1 belongs to the highly conserved cyclin family, more specifically to the cyclin D subfamily (Lew D J, Dulic V, Reed S I (1991). Isolation of three novel human cyclins by rescue of G1 cyclin (Cln) function in yeast. Cell 66, 1197-1206; Xiong Y, Connolly T, Futcher B, Beach D (1991). Human D-type cyclin. Cell 65, 691-699). Cyclins function as regulators of cyclin-dependent kinases (CDKs). Different cyclins exhibit distinct expression and degradation patterns which contribute to the temporal coordination of each event in cell cycle (Deshpande A, Sicinski P, Hinds P W (2005). Cyclins and cdks in development and cancer: a perspective. Oncogene 24, 2909-2915.) Cyclin D1 forms a complex with and functions as a regulatory subunit of CDK4 or CDK6, whose activity is required for cell cycle G1/S transition. CCND1 forms a serine/threonine kinase holoenzyme complex with CDK4 and CDK6 imparting substrate specificity to the complex (Bates S, Bonetta L, MacAllan D, Parry D, Holder A, Dickson C, Peters G (1994). CDK6 (PLSTIRE) and CDK4 (PSK-J3) are a distinct subset of the cyclin-dependent kinases that associate with cyclin D1. Oncogene 9, 71-79). Mutations, amplifications and overexpression of this gene, which alters cell cycle progression, are observed frequently in a variety of tumors and may contribute to tumorigenesis (Hedberg Y, Davoodi E, Roos G, Ljungberg B, Landberg G (1999). Cyclin-D1 expression in human renal-cell carcinoma. Int. J. Cancer 84, 268-272; Vasef M A, Brynes R K, Sturm M, Bromley C, Robinson R A (1999). Expression of cyclin D1 in parathyroid carcinomas, adenomas, and hyperplasias: a paraffin immunohistochemical study. Mod. Pathol. 12, 412-416; Troussard X, vet-Loiseau H, Macro M, Mellerin M P, Malet M, Roussel M, Sola B (2000). Cyclin D1 ex-pression in patients with multiple myeloma. Hematol. J. 1, 181-185). Cyclin D1 is overexpressed in colorectal, gastric, esophageal, lung, kidney and breast cancer, as well as leukemia and lymphoma, with little expression in normal tissue. It is also typically overexpressed in mantle cell lymphoma, which is characterized by a t(11;14)(q13;q32) translocation that juxtaposes the protooncogene CCND1 at chromosome 11q13 to the Ig heavy chain gene at chromosome 14q32 (Wang et al., 2009; Kondo et al., 2008).
A common A/G single nucleotide polymorphism (A870G) results in two distinct mRNA isoforms a and b in cyclin D1. The alternately spliced isoform b encodes a truncated protein which has been linked to higher incidence of tumor onset including sporadic RCC, lung cancer, colon cancer, and other cancer types (Fu M, Wang C, LI Z, Sakamaki T, Pestell R G (2004). Minireview: Cyclin D1: normal and abnormal functions. Endocrinology 145, 5439-5447; Yu J, Habuchi T, Tsuchiya N, Nakamura E, Kakinuma H, Horikawa Y, Inoue T, Ogawa O, Kato T (2004). Association of the cyclin D1 gene G870A polymorphism with susceptibility to sporadic renal cell carcinoma. J Urol. 172, 2410-2413). Enhanced CCND1 expression has been linked to higher tumor grades, metastasis, and decreased survival (Maeda K, Chung Y, Kang S, Ogawa M, Onoda N, Nishiguchi Y, Ikehara T, Nakata B, Okuno M, Sowa M (1998). Cyclin D1 overexpression and prognosis in colorectal adenocarcinoma. Oncology 55, 145-151; McKay J A, Douglas J J, Ross V G, Curran S, Murray G I, Cassidy J, McLeod H L (2000). Cyclin D1 protein expression and gene polymorphism in colorectal cancer. Aberdeen Colorectal Initiative. Int. J. Cancer 88, 77-81; Bahnassy A A, Zekri A R, El-Houssini S, El-Shehaby A M, Mahmoud M R, Abdallah S, El-Serafi M (2004). Cyclin A and cyclin D1 as significant prognostic markers in colorectal cancer patients. BMC. Gastroenterol. 4, 22; Balcerczak E, Pasz-Walczak G, Kumor P, Panczyk M, Kordek R, Wierzbicki R, Mirowski M (2005). Cyclin D1 protein and CCND1 gene expression in colorectal cancer. Eur. J. Surg. Oncol. 31, 721-726).
For colorectal cancer, overexpression of CCND1 at the mRNA and protein levels has been described frequently. This can be explained by the well-established fact that CCND1 is a target gene of the β-Catenin-TCF/LEF pathway which is frequently upregulated in colorectal carcinoma (Shtutman M, Zhurinsky J, Simcha I, Albanese C, D'Amico M, Pestell R, Ben-Ze'ev A (1999). The cyclin D1 gene is a target of the beta-catenin/LEF-1 pathway. Proc. Natl. Acad. Sci. U.S. A 96, 5522-5527; Tetsu O, McCormick F (1999). Beta-catenin regulates expression of cyclin D1 in colon carcinoma cells. Nature 398, 422-426).
The cyclin peptide CCN-001 (LLGATCMFV) (SEQ ID NO: 1) was identified by Sadovnikova and colleagues (Sadovnikova E, Jopling L A, Soo K S, Stauss H J (1998). Generation of human tumor-reactive cytotoxic T cells against peptides presented by non-self HLA class I molecules. Eur. J. Immunol. 28, 193-200). After induction of allo-restricted T cells (i.e. of HLA-A2-negative donors) against several Cyclin D1-derived HLA-A*02-binding peptides, it has been shown that T cell clones recognizing CCN-001 could lyse HLA-A*02 positive tumor cells endogenously expressing Cyclin D1 but not HLA-A*02 positive cells negative for Cyclin D1. Thus it was shown that CCN-001 is naturally processed and presented only by indirect evidence.
For some time it had been suggested that T cells recognizing cyclin D1-derived epitopes were absent from the T cell repertoire because of the thymic expression of cyclin D1. Assuming this, the generation of autologous CTL to cyclin D1 would have been impossible.
However, Kondo et al. (Kondo E, Maecker B, Weihrauch M R, Wickenhauser C, Zeng W, Nadler L M, Schultze J L, von Bergwelt-Baildon M S (2008). Cyclin D1-specific cytotoxic T lymphocytes are present in the repertoire of cancer patients: implications for cancer immunotherapy. Clin Cancer Res 14, 6574-6579) showed that CTL specific for the epitope CCN-001 (LLGATCMFV) could be generated from HLA-A2+ donors as well. They demonstrated this for healthy donors as well as for colon cancer patients and mantle cell lymphoma patients. As APCs, they used autologous CD40-activated B cells.
Wang et al. (Wang M, Sun L, Qian J, Han X, Zhang L, Lin P, Cai Z, Yi Q (2009). Cyclin D1 as a universally expressed mantle cell lymphoma-associated tumor antigen for immunotherapy. Leukemia 23, 1320-1328) generated cyclin D1-specific CTL against CCN-001 using T cells from mantle cell lymphoma patients and autologous mature monocyte-derived DCs as APCs. They did not use the original peptide, but a heteroclitic peptide where the first amino acid (L) was replaced by Y, enhancing the MHC binding. Resulting CTLs could kill cyclin D1+ expressing cells, including primary lymphoma cells from HLA-A2+ mantle cell lymphoma patients. The peptide was also used in other studies (Kondo E, Gryschok L, Klein-Gonzalez N, Rademacher S, Weihrauch M R, Liebig T, Shimabukuro-Vornhagen A, Kochanek M, Draube A, von Bergwelt-Baildon MS (2009a). CD40-activated B cells can be generated in high number and purity in cancer patients: analysis of immunogenicity and homing potential. Clin Exp. Immunol. 155, 249-256; Kondo E, Gryschok L, Schultze J L, von Bergwelt-Baildon M S (2009b). Using CD40-activated B cells to efficiently identify epitopes of tumor antigens. J Immunother. 32, 157-160) to demonstrate the usefulness of CD40-activated B cells as APCs. Buchner et al (in Buchner et al. Phase 1 Trial of Allogeneic Gene-Modified Tumor Cell Vaccine RCC-26/CD80/IL-2 in Patients with Metastatic Renal Cell Carcinoma. Human Gene Therapy. Mar. 2010, 21(3): 285-297) mention the CCN-001 peptide in a clinical study.
WO 2005/035714 describes vaccines for treating or preventing cancer, comprising tumor-associated HLA-restricted antigens, and in particular HLA-A2 restricted antigens. In specific aspects, cyclin D peptides are provided. Such peptides can be used to elicit specific CTLs that preferentially attack tumor cells. The cyclin D peptide may comprise the sequence LLGATCMFV, or a fragment thereof. Further described is a method for treating or prevent-ing a cancer in a patient comprising administering to the patient a therapeutically effective amount of a vaccine comprising a peptide. The method may further comprise treating the patient with a second anticancer agent, wherein the second anticancer agent is selected from a long list of agents, such as chemotherapeutic agents, such as, for example cyclophosphamide. The second anticancer agent may be administered simultaneously with the vaccine, or administered at a different time than the vaccine. Nevertheless, WO 2005/035714 does not describe any advantages regarding a combination treatment using cyclin D1 peptides.
In view of the above, very little is known about an effective use of cyclin D1 derived peptides in an effective immunotherapy against cancer. It is therefore an object of the present invention, to provide novel approaches for a more effective immunotherapeutic treatment of cancer, based on cyclin D1 derived peptides.